Fig. 7. LCN2 neurotoxicity depends on p38 MAPK and microglia.

(A) Predicted interactions between components of the gene networks shown in (Fig. 6B) and p38 MAPK. IPA software identified direct (solid lines) and indirect interactions (dashed lines). (B) Protein was extracted from cerebral cortices of 9–10 months old animals and 25 μg per sample were used for Western blotting as described in Materials and Methods. ImageJ software was used for densitometry analysis of proteins. (C) Quantification of phosphorylated p38 MAPK (pp38)/Total p38. Data was normalized to their corresponding tubulin signal. Values in graphs are mean ± s.e.m.; n = 6 (3 males and 3 females) per genotype; * P ≤ 0.05 for difference to WT control, ANOVA followed by Fisher’s PLSD post hoc test. (D-H) Rat mixed neuronal-glial cerebrocortical cell cultures (RCC) were pre-incubated with or without a p38 MAPK inhibitor, SB203580 (SB; 10 μM), for 15 min prior to treatment with LCN2 (4 nM) for 72 hrs. (D, F, and H) RCC were treated with LME (7.5 mM) to deplete microglia for 16 hrs and then pre-incubated with or without a p38 MAPK inhibitor and treated with LCN2 as described above. (E-F) Fluorescence intensity of immuno-labeled MAP2 in fixed cells was used to analyze neuronal injury and loss as described in Materials and Methods. (H-G) % Neuronal Survival was calculated by counting MAP2⁺ neurons and total cells, and calculating the percentage of neurons relative to control. Control samples were defined as 100% survival. Values in graphs are mean ± s.e.m.; n = 3 independent experiments; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, P ≤ 0.0001, ANOVA followed by Fisher’s PLSD post hoc test; scale bar = 100 μm.